The invention relates generally to the field of recombinant protein production, and particularly to the production of telopeptide collagen in recombinant host cells.
Collagen is the major protein component of bone, cartilage, skin and connective tissue in animals. Collagen in its native form is typically a rigid, rod-shaped molecule approximately 300 nm long and 1.5 nm in diameter. It is composed of three collagen polypeptide monomers which form a triple helix. Mature collagen monomers are characterized by a long midsection having the repeating sequence xe2x80x94Gly-X-Y, where X and Y are often proline or hydroxyproline, bounded at each end by the xe2x80x9ctelopeptidexe2x80x9d regions, which constitute less than about 5% of the molecule. The telopeptide regions of the chains are typically responsible for the crosslinking between the chains (i. e., the formation of collagen fibrils), and for the immunogenicity of the protein. Collagen occurs naturally in a number of xe2x80x9ctypesxe2x80x9d, each having different physical properties. The most abundant types in mammals and birds are types I, II and III.
Mature collagen is formed by the association of three procollagen monomers which include xe2x80x9cproxe2x80x9d domains at the amino and carboxy terminal ends of the polypeptides. The pro domains are cleaved from the assembled procollagen trimer to create mature, or xe2x80x9ctelopeptidexe2x80x9d collagen. The telopeptide domains may be removed by chemical or enzymatic means to create xe2x80x9catelopeptidexe2x80x9d collagen.
Interestingly, although there are a large number of different genes encoding for different procollagen monomers, only particular combinations are produced naturally. For example, skin fibroblasts synthesize 10 different procollagen monomers (proxcex11(I), proxcex11(III), proxcex11(V), proxcex12(I), proxcex12(V), proxcex13(V), proxcex11(VI), proxcex12(VI), proxcex13(VI) and proxcex11(VII)), but only 5 types of mature collagen are produced (types I, III, V, VI and VII).
Collagen has been utilized extensively in biological research as a substrate for in vitro cell culture. It has also been widely used as a component of biocompatible materials for use in prosthetic implants, sustained drug release matrices, artificial skin, and wound dressing and wound healing matrices.
Historically, collagen has been isolated from natural sources, such as bovine hide, cartilage or bones, and rat tails. Bones are usually dried, defatted, crushed, and demineralized to extract collagen, while cartilage and hide are typically minced and digested with proteolytic enzymes other than collagenase. As collagen is resistant to most proteolytic enzymes (except collagenase), this procedure can conveniently remove most of the contaminating protein that would otherwise be extracted along with the collagen. However, for medical use, species-matched collagen (e.g., human collagen for use in human subjects) is highly desirable in order to minimize the potential for immune response to the collagen material.
Human collagen may be purified from human sources such human placenta (see, for example, U.S. Pat. Nos. 5,002,071 and 5,428,022). Of course, the source material for human collagen is limited in supply and carries with it the risk of contamination by pathogens such as hepatitis virus and human immunodeficiency virus (HIV). Additionally, the material recovered from placenta is biased as to type and not entirely homogenous.
Collagen may also be produced by recombinant methods. For example, International Patent Application No. WO 97/14431 discloses methods for recombinant production of procollagen in yeast cells and U.S. Pat. No. 5,593,859 discloses the expression of procollagen genes in a variety of cell types. In general, the recombinant production of collagen requires a cloned DNA sequence encoding the appropriate procollagen monomer(s). The procollagen gene(s) is cloned into a vector containing the appropriate DNA sequences and signals for expression of the gene and the construct is introduced into the host cells. Optionally, genes expressing a prolyl-4-hydroxylase alpha subunit and a protein disulfide isomerase are also introduced into the host cells (these are the two subunits which make up prolyl-4-hydroxylase). Addition of the prolyl-4-hydroxylase leads to the conversion of some of the prolyl residues in the procollagen chains to hydroxyproline, which stabilize the triple helix and increase the thermal stability of the protein.
Alternately, recombinant collagen may be produced using transgenic technology. Constructs containing the desired collagen gene linked to the appropriate promoter/enhancer elements and processing signals are introduced into embryo cells by the formation of ES cell chimera, direct injection into oocytes, or any other appropriate technique. Transgenic production of recombinant collagen is particularly advantageous when the collagen is expressed in milk (i.e., by mammary cells), such as described in U.S. Pat. No. 5,667,839 to Berg. However, the production of transgenic animals for commercial production of collagen is a long and expensive process.
One difficulty of recombinant expression of collagen is the processing of the xe2x80x9cproxe2x80x9d regions of procollagen monomers. It is widely accepted that folding of the three monomers to form the trimer begins in the carboxyl pro-region (xe2x80x9cC propeptidexe2x80x9d) and that the C propeptide contains signals responsible for monomer selection (Bachinger et al., 1980, Eur. J Biochem., 106:619-632; Bachinger et al., 1981, J. Biol. Chem. 256:13193-13199). One group has identified a region in the carboxy pro-region that they believe is necessary and sufficient for monomer selection (Bulleid et al., 1997, EMBO J. 16(22):6694-6701; Lees et al., 1997, EMBO J. 16(5):908-916; International Patent Application No. WO 97/0831 1; McLaughling et al., 1998, Matrix Biol. 16:369-377). Additionally, Lee et al. (1992, J. Biol. Chem. 267(33):24126-24133) have shown that deletion of the N propeptide results in decreased secretion of human xcex11 pC collagen from CHL cells, but not Mov-13 cells. Accordingly, it is believed that the pro-regions must be retained for proper chain selection, alignment and folding of collagen produced by recombinant methods. In cells which normally produce collagens, specific proteolytic processing enzymes are produced which remove the N and C propeptides following the secretion of collagen. These enzymes are not present in cells which do not normally produce procollagen (including commonly used recombinant host cells such as bacteria and yeast).
Ideally, the recombinant production of collagen is accomplished with a recombinant host cell system that has a high capacity and a relatively low cost (such as bacteria or yeast). Because bacteria and yeast do not normally produce the enzyme necessary for processing of the N and C propeptides, the propeptides must be removed after recovering the recombinant procollagen from the host cells. This can be accomplished by the use of pepsin or other proteolytic enzymes such as PRONASE(copyright) or trypsin, but in vitro processing produces xe2x80x9craggedxe2x80x9d ends that do not correspond to the ends of mature collagen secreted by mammalian cells which normally produce fibrillar collagen. Alternately, the enzymes which process the N and C propeptides can be produced and used to remove the propeptides. Any contamination of these enzyme preparations with other proteases will result in ragged ends. This added processing step increases the cost and decreases the convenience of production in these otherwise desirable host cell systems.
Gelatin can be considered a collagen derivative. Gelatin is denatured collagen, generally in monomeric form, which may be fragmented as well. Gelatin serves a large number of uses, particularly in foodstuffs as well as in medicine, where it is frequently used for coating tablets or for making capsules. However, the possibility of the spread of prion-based diseases through animal-derived gelatin has made the use of animal-derived gelatin less attractive.
Accordingly, there is a need in the art for simplified methods of producing gelatin and genuine telopeptide collagen in high capacity systems.
The inventors have discovered new methods for the recombinant production of fibrillar collagens. The inventors have surprisingly and unexpectedly found that co-expression of DNA constructs encoding xcex11(I) and xcex12(I) collagen monomers lacking the N and C propeptides form heterotrimeric telopeptide collagen having the properties of genuine human type I collagen. Additionally, co-expression in yeast of DNA constructs encoding a non-collagen signal sequence linked to xcex11(I) and xcex12(I) collagen monomers lacking the N, the C, or both the N and C propeptides results in a surprising increase in the production of type I collagen. Further, the inventors have found that the efficient production of triple helical fibrillar collagen in accordance with the invention is not dependent on hydroxylation of the collagen monomers.
The methods of the instant invention may be used to produce any of the fibrillar collagens (e.g., types I-III, V and XI), as well as the corresponding types of gelatin, from any species, but are particularly useful for the production of recombinant human collagens for use in medical applications. Collagen produced in accordance with the invention may be hydroxylated (i.e., proline residues altered to hydroxyproline by the action of prolyl-4-hydroxylase) or non-hydroxylated. Additionally, the methods of the invention also provide efficient methods for production of recombinant gelatin.
In one embodiment, the invention relates to methods for producing fibrillar collagen by culturing a recombinant host cell comprising a DNA encoding a fibrillar collagen monomer lacking a C propeptide sequence selection and alignment domain (SSAD) under conditions appropriate for expression of said DNA; and producing fibrillar collagen. The DNA may encode any of the fibrillar collagen monomers, such as xcex11(I), xcex12(I), xcex11(II), xcex11(III), xcex11(V), xcex12(V), xcex13(V), xcex11(XI), xcex12(XI), and xcex13(XI). Optionally, the DNA encoding the fibrillar collagen monomer lacking a C propeptide SSAD may also lack DNA encoding the N propeptide.
In another embodiment, the invention relates to methods for producing fibrillar collagen by culturing a recombinant yeast host cell comprising a DNA encoding a fibrillar collagen monomer lacking a N propeptide under conditions appropriate for expression of said DNA; and producing fibrillar collagen.
Another embodiment relates to recombinant host cells comprising an expression construct comprising a DNA encoding a fibrillar collagen monomer lacking a C propeptide sequence selection and alignment domain (SSAD). The DNA may encode any of the fibrillar collagen monomers, such as xcex11(I), xcex12(I), xcex11(II), xcex11(III), xcex11(V), xcex12(V), xcex13(V), xcex11(XI), xcex12(XI), and xcex13(XI). Optionally, the DNA encoding the fibrillar collagen monomer lacking a C propeptide SSAD may also lack DNA encoding the N propeptide.
In a further embodiment, the invention relates to trimeric collagen molecules which lack propeptide domains and lack native glycosylation and trimeric collagen molecules which lack propeptide domains and lack any glycosylation. The trimeric collagens of the invention have xe2x80x9cgenuinexe2x80x9d ends (i.e., the amino and carboxy-terminal residues which would be produced by normal processing in tissues which naturally produce collagen).
Another embodiment of the invention relates to the production of recombinant gelatin. Gelatin may be produced using constructs encoding any collagen monomer, preferably lacking the C propeptide domain and/or the N propeptide domain in a recombinant host cell. The collagen monomers thus produced may be hydroxylated (e.g., produced in a cell with prolyl-4-hydroxylase activity) or non-hydroxylated. After collection and any purification, the collagen monomers are denatured as necessary to form gelatin, although non-hydroxylated collagen monomers expressed in host cells incubated at elevated temperatures may not require any further treatment to form gelatin.